The vast majority of neural circuit studies neglect to take into account the non-neuronal cells in the brain, but in order to truly appreciate neural circuit function, we will need to monitor and manipulate activity in many cell types. Our understanding of astrocyte signaling is years behind that of neurons, because the appropriate tools have been lacking for these largely electrically silent cells. We don't know what extracellular signals astrocytes respond to, nor how they contribute to circuit function. This is due, in part, to the lack of methods that replicate or report the breadth of possible presynaptic activity in vivo, i.e. the release of neurotransmitter. The current proposal addresses gaps in our understanding of astrocytes in neural circuit function and harnesses the power of novel optical tools to tackle them. We propose to apply a suite of fluorescent nanosensors in vivo that allow precise spatiotemporal reporting of endogenous physiological neuromodulator concentration. In Aim 1, we validate a suite of neuromodulator-specific fluorescent nanosensors with a series of both positive and negative controls to test for specificity, stability, toxicity, spatiotemporal responsivity, and two-photon excitation/emission of the nanosensors in the mouse cortex in vivo. To do so, we will use optogenetic activation of neuromodulatory input fibers to precisely release transmitter, known pharmacological manipulations, whole-cell patch- clamp electrophysiology, and population neuronal imaging. In these validation experiments, we will record the responses of the nanosensors after a well-defined manipulation to ensure accurate reporting, and confirm that neural circuit physiology is unchanged by the application of each fluorescent nanosensor. In Aim 2, we will simultaneously image extracellular neuromodulatory dynamics and intracellular astrocytic Ca2+ activity. Dual-color image analysis will probe for relationships between each transmitter and astrocyte activity in vivo.